African trypanosomes are parasitic protozoa which are responsible for human and animal diseases. They inhabit their host's bloodstream, and they evade immune defenses by antigenic variation. Antigenic variation is mediated by the variant surface glycoprotein (VSG), which forms a coat covering the entire cell. During antigenic variation, the parasite synthesizes a new VSG, with a different amino acid sequence, exchanging its old coat for a new one. Trypanosomes have the genetic potential to synthesize hundreds of different VSG molecules, and therefore they can maintain a chronic infection. This research involves the biochemistry of VSG, focusing particularly on the myristate-containing glycosyl phosphatidylinositol (GPI) which anchors it to the plasma membrane. The GPI is synthesized as a precursor, glycolipid A, which is linked to the C-terminus of the newly-made VSG. Previous studies, using a cell free system, have revealed the pathway for biosynthesis of glycolipid A and the remodeling mechanism by which myristate is incorporated into this structure. Proposed studies consist of further analysis of the mechanism by which myristate is incorporated into the GPI and also the mechanism for incorporation of its phosphoethanolamine. Other studies will focus on trypanosome myristate metabolism, with special emphasis on mechanisms of myristate uptake and intracellular transport to the site of GPI biosynthesis. There will be additional investigations of myristate analogs which are selectively toxic to African trypanosomes, possible because they interfere with VSG biosynthesis or function. Earlier studies on GPI biosynthesis and myristoylation have involved crude membrane fractions; however, a more comprehensive understanding of these processes will require enzyme purification. Purification will also allow cloning of the genes regulating GPI biosynthesis and myristoylation, and new genetic techniques will allow specific mutation of these genes in trypanosomes. This genetic analysis will allow biological studies of the effect of defective GPI biosynthesis or myristoylation. The same genetic approach will be applied to GPI-specific phospholipase C (GPI-PLC), an enzyme which cleaves the VSG anchor.
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